.beta.-glucans, homopolymers of glucose, are an abundant class of polysaccharides that include cellulose, and appear to serve structural, functional and morphological roles at the cell surface of fungi, bacteria and plants. Despite their widespread occurrence, there has been surprisingly little work to address the basis of cell wall glucan biosynthesis at the genetic and molecular level in eukaryotes. `In vitro` enzymatic reactions resulting in glucan synthesis have been defined and partially characterized for several systems though components of the synthetic machinery have eluded purification. The isolation of mutants defective in the production of cell wall glucan should define genes which encode biosynthetic enzymes as well as other products, for example those which regulate glucan synthesis or generate glucan precursors.
Mixed linked .beta.-D-glucans consisting of glucopyranosyl residues joined through (1.fwdarw.3) and (1.fwdarw.6)-linkages are common to fungi belonging to the Ascomycetes, Basidomycetes and Oomycetes. Fractionation studies of the Saccharomyces cerevisiae cell wall demonstrated the presence of several glucan subclasses, which could be structurally distinguished by polymer length and the ratio of (1.fwdarw.3) to (1.fwdarw.6)-.beta.-D-linkages. Much of the yeast cell wall glucan is isolated from whole cells as an alkali insoluble fraction which was found to contain two distinct types of polymers. The most abundant alkali insoluble glucan consists predominantly of repeating units of linear (1.fwdarw.3)-.beta.-linked residues, 3% of which are branched through a (1.fwdarw.6)-.beta.-linkage (Manners, D. J. et al., 1973, Biochem. J., 135:19-30). This glucan has a degree of polymerization estimated to be about 1,500 and may determine the shape and stability of the yeast cell wall. The other alkali insoluble glucan has a degree of polymerization estimated to be about 140 and contains residues which are predominantly connected through linear (1.fwdarw.6)-.beta.-linkages (Manners, D. J. et al., 1973, Biochem. J., 135:31-36). This glucan will be referred to as (1.fwdarw.6)-.beta.-glucan although in addition to linear (1.fwdarw.6) linked units, it is composed of some linear (1.fwdarw.3)-linked residues and a relatively high proportion of (1.fwdarw.3, 1.fwdarw.6)-linked branched residues (14%). Yeast (1.fwdarw.6)-.beta.-glucan accounts for approximately 20% of the alkali insoluble glucan or 3% of the total cellular dry weight.
The K1 killer toxin of S. cerevisiae provides a selection scheme for the isolation of mutants defective in (1.fwdarw.6)-.beta.-D-glucan production. This toxin is a protein secreted by killer yeast strains which kills sensitive (nonkiller) strains. K1 toxin displays a lectin-like affinity for linear (1.fwdarw.6)-.beta.-D-glucan and must bind to the wall of sensitive yeast in order to initiate the killing process. Mutations in the so called KRE1 gene result in killer toxin resistance, and are associated with an abnormal production of cell wall (1.fwdarw.6)-.beta.-glucan (Hutchins K. and Bussey, H., 1983, J. Bacteriol., 154:161-169).
The KRE1 gene encodes a protein directed into the yeast secretory pathway. The (1.fwdarw.6)-.beta.-glucan fraction which remained in a KRE1 mutant yeast strain had an altered structure with a smaller average polymer size, suggesting that (1.fwdarw.6)-.beta.-glucan is synthesized in a stepwise manner.
Spiros J. et al. have described methods to produce glucan particles from Saccharomyces cerevisiae strains (U.S. Pat. No. 4,810,646 issued on Mar. 7, 1989). They have also demonstrated that there is a variation in viscosity profiles of yeast glucan depending upon the strain of Saccharomyces cerevisiae used. They used the following strains of S. cerevisiae A364A, 374, 377 and a mutant R4 which is characterized by its resistance to laminarinase. These methods to produce glucans from these strains are unrelated to the present invention.
At first, the mutant R4 appears to be related to the kre mutants of the present invention and are said to have an increased .beta.-(1.fwdarw.6) glucan fraction. Glucan obtained from this mutant was shown to have an altered network-compression modulus versus volume fraction. On this basis, this mutant is said to give glucan matrices with altered structural properties, and in this general respect resembles the mutants defective in genes described in the application.
The mutant R4, although not characterised or described in detail, appears to be unrelated in properties to the kre mutants described in the present invention. The mutant R4 is said to have increased .beta.-(1.fwdarw.6) glucan production, whereas we show that kre mutants have less. Further, the mutant R4 has resistance to laminarinase, while the kre mutants in contrast show an increased sensitivity to a functionally similar (1.fwdarw.3)-.beta.-glucanase enzyme, zymolyase. In these respects, mutant R4 even though structurally altered in glucan, has different properties and characteristics from the kre mutants. The DNA sequence of the mutant R4 is not known. By not knowing the mutant R4 DNA sequence, one cannot study its mechanism of action and one is very limited in its application.
It would be highly desirable to know which gene products are required for fungal cell wall biosynthesis. These could be used as potential targets for the screening for specific antifungal agents which act against fungi pathogenic to plants and animals, including man. It would be of a great advantage to be able to selectively inhibit yeast cell growth without affecting mammalian or plant cells.
It would also be desirable to have the complete nucleic acid sequence of genes involved in the cell wall .beta.-glucan assembly pathway. Such genes could, through recombinant DNA technology, be used to overproduce glucans, to produce modified glucans, and to produce microorganisms having modified cell walls to facilitate cell lysis for extraction of proteins and other molecules.